CNS pharmacotherapy is impeded by the existence ‘CNS barriers’ at the interface between blood and neural tissue. As a result, many drugs fail as therapeutic agents for the CNS because they are pumped out of the brain. To overcome these drug efflux transporters, recent research in the field aims at identifying the factors and the intracellular signaling mechanisms implicated in their regulation in order to modulate their activity and improve pharmacotherapy of brain diseases. For years, BBB efflux transporters have been studied in the adult organism. But, there is a wide-spread belief among pediatricians, neurologists, and neuroscientists that the BBB in the embryo, fetus, newborn, and infant is ‘immature’, implying caution in giving drugs to infants . Moreover, intricate developmental processes are taking place during the prenatal and postnatal periods, which might mean that BBB efflux transporters could also undergo important changes during brain maturation, and might possibly have age-related differences in the inflammatory response. There is growing evidence suggesting that the immune system, through systemic or cerebral inflammation, disturbs the BBB efflux transporters , and these alterations can affect the efficacy of CNS-acting drugs.
However, current knowledge on the functional status of the BBB in immature organism remains very limited. Thus, from the clinical perspective of developing new drugs with enhanced efficacy in both the adult and children CNS, it is important to understand 1) the role of the BBB drug efflux transporters in the CNS at the different stages of brain maturation and 2) the mechanisms that regulate their functional activity, under both normal and inflammatory conditions.
In this study, we evaluated the impact of acute cerebral inflammation mediated by ET-1 on BBB efflux transporters with a comparison between juvenile and adult rats. Specifically, we evaluated, ex vivo and in vivo, the impact of intracerebroventricular (icv) injection of ET-1, on the expression and activity of two of the most clinically relevant BBB drug efflux transporters, the P-gp and the BCRP transporters.
Our results showed that, under inflammatory conditions, BBB drug efflux transporters are regulated differently in juvenile rats when compared to adult rats, and that this differential regulation may be due, in part, to a maturational difference in glial basal levels, and neuro-inflammatory response triggered by ET-1.
Since inflammation has been reported to influence BBB integrity [27, 28], we first investigated in our experimental conditions whether icv injection of ET-1 changed BBB permeability. BBB permeability was evaluated by assessing the brain uptake of Evans blue dye to ensure the functionality of the BBB to macromolecule entry after ET-1 treatment, and by following the expression of tight junctions on the brain microvessels to evaluate the integrity of tight junctions which restrict paracellular movement to small molecules across the BBB. Results depicted in Figure 1 demonstrate a lack of BBB permeability changes with the use of Evan blue as well as the use of baclofen, as small molecule which does not cross the BBB (data not shown). In addition, we found no modification in the ZO-1 gene expression during the cerebral inflammation triggered by ET-1 treatment, and this in both juvenile and adult brains. These findings allowed us to determine the impact of neuroinflammation on the expression and transport activity of BCRP and P-gp in adult and juvenile brains at the BBB. We first compared P-gp and BCRP transport activity between juvenile and adult BBB, and found that juvenile and adult BBB have the same P-gp and BCRP transport activity (Additional file 1). We also found evidence that the transport activities of P-gp and BCRP are enhanced by icv ET-1 at the adult BBB, whereas no significant modulation of P-gp transport activity and a tendency to decrease in BCRP transport activity were seen at the juvenile BBB (Figures 4 and 5). Thus, these results gave evidence that P-gp and BCRP transporters at the BBB level are regulated differently under pathological conditions in juvenile brain when compared to adult. These findings emphasize the importance of considering differential P-gp and BCRP transporter regulation mechanisms between juvenile and adult BBB in the context of pathological conditions.
Second, after assessing BBB transport activity in adult and juvenile brains in the context of ET-1 treatment, we aimed at understanding the underlying mechanisms behind this differential regulation. We suspected a role of neuroinflammatory response triggered by ET-1 because it is likely that cytokine secretion in adult and juvenile brains controls the regulation of BBB transporters . In the brain, the inflammatory response begins with recruitment of the innate immune system. Rapidly, in response to infection or injury, microglia, major inflammatory cells of the monocyte/microphage lineage that reside in the brain, are activated . Microglia are important phagocytic cells, and once activated they release numerous inflammatory molecules, particularly pro and anti-inflammatory cytokines and chemokines  (Proinflammatory molecules such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6) [48–50], chemokines (IL-8, MIP-1α, MIP-1β, MCP-1) [51, 52], proteases , and anti-inflammatory molecules such as TGF-β and IL-10 ). Later, astrocytes are activated . Apart from being involved in a variety of physiologic processes, astrocytes rapidly react to different neurological insults. Upon activation, a series of changes occur in astrocytes, leading to the acquisition of macrophage differentiation markers and effector properties. One main feature of these changes is the increase in the number and size of glial fibrillary acidic protein (GFAP) expressing cells. GFAP is an intermediate filament cytoskeletal protein expressed primarily by astrocytes and it is considered as the marker of astrocytes . Concomitant with GFAP overexpression, astrocytes release many proinflammatory mediators and upregulate the expression of several inflammatory molecules, contributing to the amplification of inflammation . These facts allow us in this study, to test whether the inflammatory response triggered by ET-1 can be responsible for the differential regulation of P-gp and BCRP transporters between juveniles and adults. For that, we quantified 1) the glial activation marker, the GFAP in hippocampus and cortex of rat brains treated with ET-1, and 2) a panel of cytokines and chemokines that could be induced by ET-1 in both juvenile and adult rat brains. We found a difference in the GFAP basal levels between the two populations either in hypocampus or in the brain cortex, but our results showed a pronounced glial activation in adult and juvenile cortex and hippocampus coincided with cytokine/chemokine levels (Figures 6 and 7). Our findings were that IL6, IL-1β, CCl2/MCP-1, TIMP-1 and IL-10 increased significantly in adult brain compared with juvenile brain. These results emphasize the fact that brain development such as glial maturation is likely of paramount importance in the synthesis of specific cytokines such as IL6 for example. Indeed, IL6 is a well-known marker of glial activation  and the autocrine action of this interleukin on glial cells might account for an increase of ABC transporters at the cell surface. This increase is important in the secretion of ccl2/MCP-1 by astrocytes upon toll-like receptor 3 activation as reported recently . Thus, a differential GFAP basal level and a differential secretion of cytokines might lead to a differential regulation of ABC transporters at the BBB level. Moreover, taking into account the differential effect of ET-1 on cytokine levels in juvenile and adult brains, we suggest that the increased levels of cytokines in the adult brain and more particularly IL-6 and IL-1β may cause functional but not transcriptional regulation of P-gp and BCRP in adult BBB. Indeed, there was no modulation of protein synthesis in BCRP and P-gp in adult rat brain microvessels (Figure 5). Modulation of transport activity for P-gp in response to peripheral pain inflammation with no increase in its protein expression has been previously reported . We found no published results addressing whether cerebral inflammation as mediated by ET-1 regulates either P-gp or BCRP activity at the adult or juvenile BBB. Based on our results and the literature, we suggest that cytokine synthesis in the adult brain modulates BCRP and P-gp activity at the BBB by post-translational mechanisms such as phosphorylation and cellular localization of transporters. Inflammation regulates a number of intracellular signal transduction pathways [59–61] involved in the regulation of transporter activity. At the BBB, P-gp has been localized to plasma membrane surfaces as well as several subcellular sites, and there is overwhelming evidence suggesting that the localization of P-gp and its trafficking within brain endothelial cells contributes to its function [62, 63]. To formally demonstrate that cytokine synthesis is a cause of the differential regulation of transporter activity between juvenile and adult BBB, we increased the amount of ET-1 administered after determining the lack of BBB breakdown. At the dose of 125 pmol/kg of ET-1, the juvenile brain exhibited increases in IL1β, MCP-1, and IL6 ( Additional file 2). Despite the level of those cytokines in the brain, the juvenile BBB showed a decrease in BCRP activity which strikingly coincided with the decrease of unbound plasma prazosin concentration (3.77 ± 1.58 ng/ml versus 8.56 ± 3.77 ng/ml, P <0.05). In addition, the increase in the administered dose of ET-1 did not change the activity of P-gp in the juvenile BBB. We observed no significant decrease of digoxin concentration in unbound plasma of juvenile animals (23.50 ± 7.58 treated animals versus 47.88 ± 6.80 for control animals, P <0.05). In adult brain, BBB at the dose of 125 pmol/kg of ET-1 (data not shown) showed the same profile regarding the increase of P-gp and BCRP activity compared with the dose of 25 pmol/kg. These findings suggest the involvement of other parameters in the differential regulation of P-gp and BCRP at the BBB, particularly in juvenile brain. Thus, further investigation is warranted to define more precisely the underlying mechanisms. It has been reported that the transcriptional activity of ABC transporters is under the control of orphan nuclear receptors such as steroid and xenobiotic receptors, and that their expression and function are regulated by environmental stimuli that induce stress. Recent studies show that increased transporter expression occurs in response to signals that activate specific transcription factors including, PXR, CAR, NF-κB and AP-1, and reduced transporter activity occurs rapidly and reversibly in response to signaling through Src kinase, protein kinase C and estrogen receptors . Moreover, Bauer and colleagues have shown in rat brain capillaries that tumor necrosis factor alpha (TNF-α) binding to its receptor TNFR1 leads to the release of ET-1 which in turn acts through its receptor ETB to continue the signaling cascade via nitric oxide synthase (NOS) and protein kinase C (PKC) . This long-term exposure leads to an increase of P-gp activity  which is in agreement with our results regarding the adult brain but not the juvenile brain even at low dose of ET-1 (25 pmol/kg) or at the dose of 125 pmol/kg. Thus, to determine the underlying mechanisms behind the differential regulation of the BBB P-gp and BCRP transporter further studies are required and these must be primarily focused on the role of these multiple signaling pathways modulating the expression and activity of ABC transporters at the BBB level in children compared to adults. In addition, difference in the ontogenesis of ET-1 receptor (ETA) evidenced (data not shown) in our laboratory at the level of BBB in juvenile and adult brain might be also taken into account to explain this differential functional regulation of P-gp and BCRP transporter.